A method for perfusion of a pig kidney ex vivo and use thereof

Abstract

The application discloses a method for perfusion of an isolated pig kidney and application thereof. The method comprises using a peristaltic pump to perfuse physiological saline into the isolated pig kidney, and collecting generated urine. Compared with a static cold-preserved pig kidney, the isolated pig kidney obtained by using the perfusion method provided by the application can better maintain the integrity of a renal tubular structure according to a comparison of tissue sections after perfusion for 1 hour. The isolated pig kidney obtained by using the perfusion method provided by the application can effectively maintain the filtration function of the kidney during extracorporeal circulation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for perfusion of isolated porcine kidneys and its application. Background Technology

[0002] Mouse-based kidney models are currently the most commonly used animal models for conducting kidney-related research. They have advantages such as controllable experimental conditions, relatively simple breeding conditions, and strong reproducibility, making them highly versatile. However, the inherent biological characteristics of these models prevent them from accurately reflecting the actual situation of related research in human kidneys. The significant physiological differences between mouse and human kidneys are one of the reasons why mouse-based research cannot be successfully translated into clinical applications.

[0003] The similarities between pig kidneys and human kidneys make pig kidneys one of the most important animal sources in current xenotransplantation research. The main similarities include: similar anatomical structure, highly consistent physiological functions, similar vascular systems, high homology at the gene and molecular level, and the ability to meet the size requirements of clinical replacement organs. Therefore, research models based on pig kidneys are expected to provide scientific data that are closer to the real state of human kidneys. However, at present, research on pig kidney perfusion models lacks a standardized modeling process and pig kidney function characterization.

[0004] Therefore, there is an urgent need in this field for a perfusion method that is easy to operate, has low equipment cost, and can effectively maintain the function and vitality of isolated pig kidneys. Summary of the Invention

[0005] To address the lack of a standardized, reproducible, cost-effective, and efficient ex vivo porcine kidney perfusion technique in existing technologies, and to provide a reliable experimental model that more closely resembles the physiological state of humans for kidney-related research (especially drug toxicity evaluation and organ preservation), this invention provides a method for ex vivo porcine kidney perfusion and its application.

[0006] The present invention provides a method for perfusion of isolated porcine kidney, the method comprising perfusing physiological saline into isolated porcine kidney using a peristaltic pump and collecting the resulting urine.

[0007] In some implementations, the temperature of the physiological saline infused in the method is 0-4 degrees Celsius.

[0008] In some implementations, the peristaltic pump rotates at a speed of 55-65 rpm during infusion, for example, 60 rpm.

[0009] In some implementations, when the method uses a peristaltic pump to infuse physiological saline into isolated pig kidneys, the peristaltic pump is used in a pulse mode.

[0010] In some embodiments, the flow rate of the peristaltic pump during perfusion is 60-80 mL / min, for example, 70 mL / min.

[0011] In some implementations, the urine production rate in the method is 1-1.5 mL / min.

[0012] In some implementations, the isolated porcine kidney is a healthy kidney.

[0013] In some implementations, the surface of the isolated porcine kidney is intact, without any damage or cracks.

[0014] In some embodiments, the method uses an ex vivo kidney perfusion system for perfusion, the kidney perfusion system comprising a reservoir, a peristaltic pump, a membrane oxygenator, a heat exchanger, a control system, a pressure / flow sensor, a kidney, and connecting tubing, wherein the reservoir, peristaltic pump, cryogenic operating table, pressure / flow sensor, and kidney are sequentially connected via connecting tubing.

[0015] In some implementations, the connecting tube includes an arterial connecting tube and a venous return tube.

[0016] In some implementations, the venous return tube is also referred to as a ureter, which is inserted into the ureter of the kidney (without inserting into the renal pelvis) to collect urine separately, and a metal vascular clamp is used around the ureter to fix the ureteral tube.

[0017] In some implementations, the arterial connection tube is also known as a renal artery cannula, used to infuse physiological saline into the kidney.

[0018] In some embodiments, the peristaltic pump has a unidirectional liquid pumping capability, can withstand resistance of 1000 mmHg, and ensures uniform and constant liquid flow under different resistances; for example, the Baoding Dichuang peristaltic pump with model number BT600S.

[0019] Another aspect of the present invention provides a method for studying drug-induced nephrotoxicity and / or drug distribution in the kidney, the method comprising collecting urine produced from isolated porcine kidneys perfused by the method provided above for testing and / or performing pathological examination on kidney tissue.

[0020] In some implementations, urine is tested by detecting AST, LDH, KIM-1, and NGAL levels.

[0021] Aspartate aminotransferase (AST) is an important transaminase that is widely found in tissues such as the liver, myocardium, skeletal muscle, and kidneys. It is an important marker of hepatocellular damage, and elevated serum AST levels usually indicate liver damage, myocardial infarction, or muscle damage.

[0022] Lactate dehydrogenase (LDH) is a key enzyme for cellular metabolic adaptation under stress and a classic biological marker of cellular structural damage. When severe stress (such as toxic stimulation, inflammation, or ischemia-reperfusion) impairs cell membrane integrity, intracellular LDH is released extracellularly. The amount of LDH released is often used as a non-specific indicator of cell damage or cell death, particularly in in vitro and tissue perfusion models.

[0023] Kidney Injury Molecule-1 (KIM-1) is a type I transmembrane glycoprotein that is almost undetectable in normal kidney tissue, but is rapidly and extensively expressed in proximal tubular epithelial cells after renal ischemia or toxic injury. It is an early and sensitive biomarker of acute kidney injury (AKI), and its upregulation usually precedes an increase in serum creatinine.

[0024] Neutrophil gelatinase-associated lipocarboxin (NGAL) is a small secretory protein expressed in various cells, including neutrophils and renal tubular epithelial cells. It is an early and sensitive marker of acute kidney injury, and elevated levels can be detected in plasma and urine within 2-3 hours after kidney injury. It can be used for early diagnosis and prognostic assessment of AKI.

[0025] The combination of AST, LDH, KIM-1 and NGAL indicators was used for dynamic assessment of isolated kidney injury, which can effectively assess the degree of kidney damage and provide an effective detection method for verifying drug-induced kidney damage.

[0026] In some implementation schemes, pathological testing is performed by tissue biopsy.

[0027] In some implementations, pathological examination is performed by tissue sections and H&E staining.

[0028] In some embodiments, the method described above for collecting urine produced from the isolated porcine kidney perfused with the method of the present invention for testing and / or performing pathological examination of the kidney tissue includes a step of assessing the function of the isolated porcine kidney using creatinine.

[0029] Another aspect of the present invention provides a method for evaluating the function of isolated porcine kidneys, the method comprising perfusing physiological saline containing creatinine into isolated porcine kidneys perfused by the method provided above, collecting the resulting urine and detecting the creatinine content, wherein the amount of creatinine added is 50-500 μmol / L.

[0030] In some embodiments, the amount of creatinine added is 100-300 μmol / L.

[0031] In some embodiments, the amount of creatinine added is 100 μmol / L.

[0032] Creatinine is a product of muscle metabolism and is primarily excreted by the kidneys through glomerular filtration and tubular secretion. The ability of pig kidneys to clear creatinine is similar to that of human kidneys; normal serum creatinine levels in pigs are approximately 0.6–1.6 mg / dL. In xenotransplantation studies, successfully transplanted pig kidneys effectively reduce serum creatinine levels in recipients. For example, in pig-human transplantation experiments, creatinine levels decreased from 3.9 mg / dL to 0.9 mg / dL, with a clearance rate reaching 200 mL / min, indicating that pig kidneys possess normal creatinine metabolism and excretion functions.

[0033] In clinical applications of xenotransplantation, serum creatinine levels are a core indicator for assessing porcine kidney function. Continuous monitoring of creatinine changes can accurately determine the survival status and functional performance of the transplanted kidney, and creatinine clearance rate directly reflects glomerular filtration function.

[0034] Another aspect of the present invention provides a method for constructing an acute kidney injury model, the method comprising adding an injury inducer to an isolated porcine kidney perfused by the method described above; the injury inducer being, for example, cisplatin or cephalosporin.

[0035] In some embodiments, the amount of cisplatin added is 0.1-2.0 μg / mL, for example 0.5 μg / mL.

[0036] In some embodiments, the amount of cephalosporin added is 1-5 μg / mL, for example 2 μg / mL.

[0037] In some implementations, the method further includes collecting the generated urine and detecting AST, LDH, KIM-1, and NGAL indicators.

[0038] Another aspect of the present invention provides a perfusion system for an isolated porcine kidney, the perfusion system comprising a reservoir, a peristaltic pump, a membrane oxygenator, a heat exchanger, a control system, a pressure / flow sensor, a kidney, and connecting tubing, wherein the reservoir, peristaltic pump, cryogenic operating platform, pressure / flow sensor, and kidney are sequentially connected via connecting tubing; it is used to perform the method of perfusion of the isolated porcine kidney provided by the present invention, or the method of nephrotoxicity and / or drug distribution in the kidney provided by the present invention, or the method of evaluating the function of the isolated porcine kidney provided by the present invention, or the method of constructing an acute kidney injury model provided by the present invention.

[0039] In some implementations, the total volume of the devices included in the infusion system is no more than 0.5 cubic meters and the weight is no more than 15 kg, making it easy to move in different locations.

[0040] Another aspect of the present invention provides a combination of biomarkers for evaluating the quality of acute kidney injury model construction, the combination of biomarkers including AST, LDH, KIM-1 and NGAL.

[0041] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0042] The reagents and raw materials used in this invention are all commercially available.

[0043] The positive and progressive effects of this invention are as follows:

[0044] Convenience: The perfusion device used in this invention is simple, compact, and easy to operate. Compared to large and complex life support systems, this device is ready to use out of the box, requires fewer connecting tubes, greatly shortens preparation and operation time, lowers the barrier to entry, and is very suitable for rapid clinical deployment and basic research.

[0045] High efficiency: Through the optimized low-temperature pulsed perfusion mode, it can effectively meet the low metabolic needs of the kidney in the isolated state, remove harmful metabolic products, and significantly prolong the preservation time of the kidney.

[0046] Dynamic functional assessment: By monitoring key parameters such as perfusion flow and vascular resistance in real time, the viability of the kidney can be objectively and quantitatively assessed, providing a reliable basis for determining whether it is suitable for transplantation or specific experiments, which cannot be achieved by static cold preservation.

[0047] Cost-effectiveness: The equipment required by this invention is simple and the cost of consumables is controllable, which is conducive to its application in medical institutions and research units with limited resources. It is especially significant for promoting the application of pig kidneys in xenotransplantation and biomedical research.

[0048] Tissue sections of isolated porcine kidneys obtained using the perfusion method provided by this invention, compared with those of porcine kidneys that were statically cryopreserved, were examined one hour after perfusion. The results showed that the method of this invention better maintained the integrity of the renal tubular structure. The glomeruli and renal tubular structures were well preserved, the renal tubular epithelium was relatively intact, with only a small amount of brush border detachment and very slight cellular edema, significantly superior to the control group kidneys that were statically cryopreserved at the same time.

[0049] The isolated porcine kidneys obtained using the perfusion method provided by this invention can effectively maintain the filtration function of the kidneys during extracorporeal circulation. Attached Figure Description

[0050] Figure 1This is a schematic diagram of the portable ex vivo kidney perfusion system described in this invention. (It includes a reservoir, a peristaltic pump, a heat exchanger, a control system, a pressure / flow sensor, a kidney, an arterial connection tube, and a venous return tube.)

[0051] Figure 2 The image shows a comparison of tissue sections (H&E staining) of a pig kidney perfused using the method of the present invention and a pig kidney that has been statically cryopreserved one hour after perfusion, demonstrating that the method of the present invention can better maintain the integrity of the renal tubular structure.

[0052] Figure 3 The urine production levels of pig kidneys under different perfusion fluid flow rates are represented, where K1-6 indicates six pig kidneys included in the study.

[0053] Figure 4 The urine volume-time curve of pig kidney after perfusion.

[0054] Figure 5 The curves showing the changes in creatinine content in perfusion fluid and urine after perfusion of pig kidneys with physiological saline containing creatinine are shown. K7-12 indicates that all 6 pig kidneys showed good renal function under this perfusion system.

[0055] Figure 6 The curves showing the changes in AST, LDH, KIM-1, and NGAL levels in the perfusion fluid and urine after perfusion of pig kidneys with cisplatin-containing saline are shown. K represents the slope of the fitted line graph, and P < 0.01 indicates that the data in this group are highly statistically significant compared with the control group.

[0056] Figure 7 The curves showing the changes in AST, LDH, KIM-1, and NGAL levels in perfusion fluid and urine after perfusion of pig kidneys with physiological saline containing cephalosporins are shown. K represents the slope of the fitted line graph, and P < 0.01 indicates that the data in this group are highly statistically significant compared with the control group.

[0057] Figure 8 The image shows H&E staining of glomeruli and tubules of pig kidneys after perfusion using the method of this invention. Detailed Implementation

[0058] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0059] Requirements for grouting tools and equipment

[0060] Peristaltic pump (Baoding Dichuang - Peristaltic Pump BT600S+YZ15; provides the primary power for liquid circulation at room temperature, has unidirectional liquid pumping capability, can withstand resistance of 1000mmHg, and ensures a uniform and constant pumping volume under different resistances; the peristaltic pump can be connected to 16-19 gauge silicone tubing; the pump's squeeze wheel should be separate from the liquid flow line, and the pump's waterproof rating should be higher than IP44); miniature pressure sensor; centrifuge tubes (50 mL, 15 mL). mL); sterile water tray (30cm×25cm); surgical instruments (curved-tip tissue scissors, straight-round tissue scissors, curved hemostats, tissue forceps, toothed forceps, No. 22 blade, vascular clamps, Jinzhong brand); No. 1 and No. 19 silicone tubing (1m in length); adapter (can connect to No. 16 and No. 19 silicone tubing, infusion tubing, Shandong Weigao); ice box (10×5cm); puncture needle; sterile saline; ELISA kits (Kim-1 and NGAL, Nanjing Boyan Biotechnology); paraformaldehyde and glutaraldehyde fixative (BioSharp); surgical sutures (2.0, 1.0 Jinzhong); round needle; exogenous creatinine (Guangzhou Zhuanyan Biotechnology Co., Ltd.); thermometer.

[0061] Porcine kidney acquisition and pretreatment

[0062] One healthy experimental miniature pig (Beijing Shichuang Century) was anesthetized, cannulated through the abdominal aorta, and irrigated with 4°C physiological saline to obtain the left kidney. The kidney weighed 120g. The pig kidney was obtained under aseptic conditions.

[0063] Place three ice boxes in a sterile water tray and add 2000 mL of pre-cooled physiological saline (0-4 degrees Celsius).

[0064] Rinse the surface of blood and dirt in a basin of water.

[0065] Large pieces of fat and renal parenchyma on the surface were bluntly separated, while preserving the fibrous membrane as intact as possible.

[0066] When separating to the renal pelvis, carefully remove excess fatty tissue with ophthalmic scissors until the anatomical layers of the renal artery, vein, and ureter are exposed.

[0067] Observe the surface of the kidney for any damage or lacerations. Lacerations within 2 cm should be closed with simple interrupted sutures using 2.0 sutures, and lacerations within 1 cm should be closed with simple interrupted sutures using 1.0 sutures. If the laceration is too large to be closed by suturing, the kidney should be abandoned for perfusion.

[0068] A ureteral catheter is inserted into the ureter (without inserting into the renal pelvis) to collect urine separately. A metal vascular clamp is used around the ureter to secure the ureteral catheter.

[0069] Insert a renal artery cannula (16 or 19 gauge silicone tubing) and slowly and evenly inject saline solution into the kidney using a syringe. Observe whether there is blood flow from the renal vein and at the same time observe whether there is a fistula on the surface of the kidney.

[0070] Sterile saline was continuously injected through the renal artery until the perfusion fluid flowing out of the renal vein became clear.

[0071] Example 1: Extracorporeal circulation protocol and functional status assessment of pig kidney

[0072] 1. Move the kidney to the peristaltic pump device described in this invention, insert an arterial cannula into the renal artery, and fix it in place.

[0073] 2. Add 1000 mL of pre-cooled sterile saline solution, place it in 3 ice boxes, and insert a water thermometer to ensure the temperature is between 0 and 4 degrees Celsius.

[0074] 3. Set the peristaltic pump to pulse mode, the speed to 60 rpm, and the flow rate to 60-120 mL / min.

[0075] 4. Perfusion continued for 1 hour, with tissue samples taken by puncture needle every ten minutes. Perfusion fluid and urine were collected simultaneously (urine volume was collected per minute in the first five minutes after starting the machine, and the perfusion fluid flow rate was adjusted to control the urine production rate at 1-1.5 mL / min. After the urine production rate was constant, 5 mL of urine sample and perfusion fluid sample were collected every ten minutes. At the same time, an equal volume of physiological saline was added to the water tray (creatinine was added if the renal filtration rate was to be studied) to maintain a constant fluid volume. Tissue samples were taken from normal tissue sites. Perfusion continued for 1 hour.

[0076] 5. After perfusion, the kidney tissue was subjected to pathological examination. Figure 8 The kidneys showed good preservation of glomerular and tubular structures, relatively intact tubular epithelium, with a small amount of brush border detachment and only very slight cellular edema, which was significantly better than the control group kidneys that were statically cryopreserved at the same time.

[0077] Tissue samples were sent to a CNAS-accredited pathology testing institution (such as the Department of Pathology at the First Hospital of Jilin University) to examine the degree of tissue damage. Results were as follows... Figure 2 As shown, a comparison of tissue sections of pig kidneys perfused using the method of the present invention and those of pig kidneys that have been statically cryopreserved one hour after perfusion shows that the method of the present invention can better maintain the integrity of the renal tubular structure.

[0078] To establish optimal perfusion flow parameters, this study progressively adjusted the peristaltic pump flow rate within the range of 60-120 mL / min and systematically observed the dynamic response of urine flow. Figure 3As shown, when the perfusion flow rate was set to 70 mL / min, the urine flow rate of each group (K2-K5) stabilized at 1-1.5 mL / min, which was closest to the physiological level and had the least variability. Based on the characteristics of stable urine production and high reproducibility at this flow rate, 70 mL / min was uniformly used as the standard perfusion condition in subsequent experiments.

[0079] To systematically evaluate the effects of different perfusion solutions on the function of isolated porcine kidneys and optimize perfusion conditions, this study used KPS (Kidney Perfusion Solution-1) for perfusion in the initial stage (0-70 min); then switched to Normal saline (0.9% sodium chloride) perfusion (80-110 min); and finally used UW (University of Wisconsin solution) perfusion (120-150 min). Figure 4 As shown, during the 80-110 min saline perfusion period, the urine flow rate remained stable at 1-1.5 mL / min. This flow rate range is closest to the physiological state, and the perfusion pressure is stable, which is beneficial for subsequent mechanism research. Therefore, saline was chosen as the perfusion fluid for subsequent experiments.

[0080] The perfusion fluid consisted of physiological saline and creatinine 133 μmol / L. The perfusion fluid and urine samples were sent to a qualified medical testing institution (such as Huace Aipu Medical Laboratory) for creatinine concentration determination. Figure 5 The results showed that after adding creatinine to the perfusion fluid, samples of the perfusion fluid and urine were taken during the perfusion process. The results proved that creatinine in the perfusion fluid was continuously filtered out by the kidneys, demonstrating that the kidneys in extracorporeal circulation have filtration function.

[0081] The perfusion fluid consisted of: normal saline and cisplatin 0.5 μg / mL; or, normal saline and cephalosporin 2 μg / mL. Kidney damage was assessed by evaluating AST, LDH, KIM-1, and NGAL levels in perfusion and urine samples collected every ten minutes. Figure 6 The results of AST, LDH, KIM-1, and NGAL tests on perfusion fluid and urine samples after perfusion with cisplatin-containing saline showed that cisplatin caused a significant increase in injury indicators. The slope of the injury indicator curves changed significantly before and after drug administration, which is consistent with clinical experience and the difference was statistically significant. Figure 7 The results of AST, LDH, KIM-1, and NGAL tests on perfusion fluid and urine samples after perfusion with cephalosporin-containing saline showed that cephalosporin did not cause significant kidney damage. In summary, this demonstrates that the renal circulation protocol constructed in this invention has the ability to respond to drug-induced damage and can also be used for the detection of kidney damage in healthy pigs.

Claims

1. A method for perfusion of isolated porcine kidney, characterized in that, The method involves using a peristaltic pump to infuse physiological saline into an isolated pig kidney and collecting the resulting urine.

2. The method as described in claim 1, characterized in that, The method satisfies one or more of the following: a. The temperature of physiological saline is 0-4 degrees Celsius; b. The peristaltic pump operates at a speed of 55-65 rpm, for example, 60 rpm; c. The flow rate for peristaltic pump infusion is 60-80 mL / min, for example, 70 mL / min; d. The rate of urine production is 1-1.5 mL / min; and, e. The isolated pig kidneys are healthy pig kidneys.

3. The method as described in claim 1 or 2, characterized in that, The method uses an ex vivo kidney perfusion system for perfusion, which includes a reservoir, a peristaltic pump, a membrane oxygenator, a heat exchanger, a control system, a pressure / flow sensor, a kidney, and connecting tubing. The reservoir, peristaltic pump, cryogenic operating table, pressure / flow sensor, and kidney are sequentially connected via connecting tubing.

4. The method according to any one of claims 1-3, characterized in that, The peristaltic pump has a unidirectional liquid pumping capability, can withstand resistance of 1000 mmHg, and ensures uniform and constant liquid flow under different resistance conditions; for example, the Baoding Dichuang peristaltic pump with model number BT600S.

5. A method for studying drug-induced nephrotoxicity and / or drug distribution in the kidneys, characterized in that, The method includes collecting urine produced from isolated porcine kidneys perfused according to any one of claims 1-4 for testing and / or performing pathological examination on kidney tissue.

6. A method for evaluating the function of isolated porcine kidneys, characterized in that, The method includes perfusing physiological saline containing creatinine into an isolated porcine kidney perfused according to any one of claims 1-4, collecting the resulting urine and detecting the creatinine content, wherein the amount of creatinine added is 50-500 μmol / L, preferably 100-300 μmol / L.

7. A method for constructing an acute kidney injury model, characterized in that, The method includes adding a damage inducer to an isolated porcine kidney perfused with the method described in any one of claims 1-4; the damage inducer is, for example, cisplatin or cephalosporin.

8. The method for constructing an acute kidney injury model as described in claim 7, characterized in that, The method also includes collecting the generated urine and detecting AST, LDH, KIM-1, and NGAL indicators.

9. A perfusion system for isolated porcine kidneys, characterized in that, The perfusion system comprises a reservoir, a peristaltic pump, a membrane oxygenator, a heat exchanger, a control system, a pressure / flow sensor, a kidney, and connecting tubing. The reservoir, peristaltic pump, cryogenic operating table, pressure / flow sensor, and kidney are sequentially connected via connecting tubing. It is used to perform the method described in any one of claims 1-4, or the method for drug-induced nephrotoxicity and / or drug distribution in the kidney as described in claim 5, or the method for evaluating isolated porcine kidney function as described in claim 6, or the method for constructing an acute kidney injury model as described in claim 7 or 8.

10. A combination of biomarkers for evaluating the quality of acute kidney injury model construction, characterized in that, The biomarker combination includes AST, LDH, KIM-1, and NGAL.

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